Naturally Occurring Enzyme Activity Variation in DROSOPHILA MELANOGASTER. II. Relationships among Enzymes

This report describes an investigation of the specificities of the genetic effects, caused by whole chromosome substitution, on the activities of 23 enzymes in Drosophila melanogaster. Two types of correlation estimates are examined, the product-moment correlation over the chromosome substitution line means and the corresponding correlation of line effects, which is a standardized covariance component estimate. The two types of correlations give very similar results. Although there is ample evidence for specific line effects on individual enzyme activities, there are extensive intercorrelations among many of the enzymes for both second- and third-chromosome substitution lines. The pattern of correlations with respect to the metabolic functions or other properties of the enzymes is difficult to visualize by inspection of the correlation matrix, so a multivariate graphical technique, the biplot (Gabriel 1971), was employed to obtain a two-dimensional view of relationships among the enzyme activities. The second and third chromosome lines show similar patterns. Four of the five mitochondrial enzymes form one highly intercorrelated group whereas another highly intercorrelated group contains several cytosolic enzymes. Within the cytosolic group, particularly high correlations are observed between enzymes that have glucose 6-phosphate as a substrate or product and between enzymes that are NADP-dependent. Although the pattern of intercorrelations is not clearly explicable in terms of metabolic relationships among the enzymes, there is some tendency for enzymes that catalyze sequential reactions or share a substrate or product to have correlated activity levels.     

Autosomal Factors with Correlated Effects on the Activities of the Glucose 6-Phosphate and 6-Phosphogluconate Dehydrogenases in DROSOPHILA MELANOGASTER

Isogenic lines, in which chromosomes sampled from natural populations of D. melanogaster are substituted into a common genetic background, were used to detect and partially characterize autosomal factors that affect the activities of the two pentose phosphate pathway enzymes, glucose 6-phosphate dehydrogenase (G6PD) and 6-phosphogluconate dehydrogenase (6PGD). The chromosome 3 effects on G6PD and 6PGD are clearly correlated; the chromosome 2 effects, which are not so great, also appear to be correlated, but the evidence in this case is not so strong. Examination of activity variation of ten other enzymes revealed that G6PD and 6PGD are not the only pair of enzymes showing a high positive correlation, but it is among the highest in both sets of lines. In addition, there was some evidence that the factor(s) affecting G6PD and 6PGD may also affect two other metabolically related enzymes, transaldolase and phosphoglucose isomerase.—Rocket immunoelectrophoresis was used to estimate specific CRM levels for three of the enzymes studied: G6PD, 6PGD and ME. This experiment shows that a large part of the activity variation is accounted for by variation in CRM level (especially for chromosome 3 lines), but there remains a significant fraction of the genetic component of activity variation that is not explained by CRM level.—These results suggest that the autosomal factors are modifiers involved in regulation of the expression of the X-linked structural genes for G6PD and 6PGD, but a role in determining part of the enzymes' primary structure cannot be excluded with the present evidence.     

Short-term heritable variation overwhelms 200 generations of mutational variance for metabolic traits in Caenorhabditis elegans

Metabolic disorders have a large heritable component, and have increased markedly in human populations over the past few generations. Genome-wide association studies of metabolic traits typically find a substantial unexplained fraction of total heritability, suggesting an important role of spontaneous mutation. An alternative explanation is that epigenetic effects contribute significantly to the heritable variation. Here, we report a study designed to quantify the cumulative effects of spontaneous mutation on adenosine metabolism in the nematode Caenorhabditis elegans, including both the activity and concentration of two metabolic enzymes and the standing pools of their associated metabolites. The only prior studies on the effects of mutation on metabolic enzyme activity, in Drosophila melanogaster, found that total enzyme activity presents a mutational target similar to that of morphological and life-history traits. However, those studies were not designed to account for short-term heritable effects. We find that the short-term heritable variance for most traits is of similar magnitude as the variance among MA lines. This result suggests that the potential heritable effects of epigenetic variation in metabolic disease warrant additional scrutiny.     

Heritabilities and additive genetic variances of the activities of some enzymes in Drosophila melanogaster populations living in different habitats

Drosophila melanogaster samples were collected from a large population in two habitats: farmyards and distilleries. Samples were taken from two villages in each habitat. Three isofemale lines were established from all four samples and full-sib crosses were set in each isofemale line. Activities of four enzymes (ADH, alpha GPDH, IDH and 6PGDH) were measured in the offspring of each cross on starch gel after electrophoresis. Broad sense heritabilities and additive genetic variances were estimated in all four samples. Most of the activity variation was observed within the isofemale lines. The isofemale lines tended to be more different in the distilleries than in the farmyards. There was no significant difference in the average activities between the two habitats for any of the enzymes investigated. The additive genetic variance of the enzyme activities did not exhibit a consistent habitat pattern. In the farmyard habitat, we detected a higher activity variation in Tiszafüred than in the other village. Strong correlation was observed among the activities of the enzymes investigated. Correlation coefficients indicated higher level of correlation in the samples collected in Tiszafüred than in those originating from Tiszaszolos. The heritability values were rather high and they had a considerable variation both between the habitats and across the enzymes.     

Genetic Analysis of Natural Populations of DROSOPHILA MELANOGASTER in Japan. III. Genetic Variability of Inducing Factors of Amylase and Fitness

"Inducibility" of amylase in Drosophila melanogaster was defined and investigated in a natural population from Japan. Inducibility represents the effects of factors remote from the structural gene that control the amount of enzyme produced. Inducibility of an isogenic line is measured as the ratio of the enzyme's specific activity in two different inducing environments. There was considerable genetic variability with respect to inducibility of amylase in 44 isogenic lines derived from a natural population of D. melanogaster . Net fitness and its components in these isogenic lines were also measured. The results indicated that, although the inducibility of the enzyme was positively correlated with the net fitness (r_g = 0.63 ± 0.2), the enzyme activities in the normal medium were not (r_g = 0.12 ± 0.37). The analysis of the data shows that the differences in inducing factors are mainly responsible for the differences in the fitness of lines and are the genetic materials for the adaptive evolution of organisms.     

Regulation of Enzyme Activities in Drosophila: Genetic Variation Affecting Induction of Glucose 6-Phosphate and 6-Phosphogluconate Dehydrogenases in Larvae

The genetic basis of modulation by dietary sucrose of the enzyme activities glucose-6-phosphate dehydrogenase (G6PD) and 6-phosphogluconate dehydrogenase (6PGD) activities in third instar larvae of Drosophila melanogaster was investigated, using isogenic lines derived from wild populations. Considerable genetically determined variation in response was detected among lines that differed only in their third chromosome constitution. Comparison of cross-reacting material between a responding and a nonresponding line showed that the G6PD activity variation is due to changes in G6PD protein level. These differences in responses are localized in the fat body, with 300 mM sucrose in the diet resulting in a sixfold stimulation of G6PD activity and a fourfold one of 6PGD in the line showing the strongest response. In this tissue, the responses of the two enzymes are closely correlated with one another. Using recombinant lines, we obtained data that suggested the existence of more than one gene on chromosome III involved in the regulation of G6PD in the fat body, and at least one of these genes affects the level of 6PGD as well.     

Genetic Variability of Flight Metabolism in DROSOPHILA MELANOGASTER . II. Relationship between Power Output and Enzyme Activity Levels

The major goal of the studies reported here was to determine the extent to which genetic variation in the activities of the enzymes participating in flight metabolism contributes to variation in the mechanical power output of the flight muscles in Drosophila melanogaster. Isogenic chromosome substitution lines were used to partition the variance of both types of quantitative trait into genetic and environmental components. The mechanical power output was estimated from the wingbeat frequency, wing amplitude and wing morphology of tethered flies by applying the aerodynamic models of Weis-Fogh and Ellington. There were three major results. (1) Chromosomes sampled from natural populations provide a large and repeatable genetic component to the variation in the activities of most of the 15 flight metabolism enzymes investigated and to the variation in the mechanical power output of the flight muscles. (2) The mechanical power output is a sensitive indicator of the rate of flight metabolism (i.e., rate of oxygen consumption during tethered flight). (3) In spite of (1) and (2), no convincing cases of individual enzyme effects on power output were detected, although the number and sign of the significant enzyme-power correlations suggests that such effects are not totally lacking.     

Genotype and sex-based host variation in behaviour and susceptibility drives population disease dynamics

Host heterogeneity in pathogen transmission is widespread and presents a major hurdle to predicting and minimizing disease outbreaks. Using Drosophila melanogaster infected with Drosophila C virus as a model system, we integrated experimental measurements of social aggregation, virus shedding, and disease-induced mortality from different genetic lines and sexes into a disease modelling framework. The experimentally measured host heterogeneity produced substantial differences in simulated disease outbreaks, providing evidence for genetic and sex-specific effects on disease dynamics at a population level. While this was true for homogeneous populations of single sex/genetic line, the genetic background or sex of the index case did not alter outbreak dynamics in simulated, heterogeneous populations. Finally, to explore the relative effects of social aggregation, viral shedding and mortality, we compared simulations where we allowed these traits to vary, as measured experimentally, to simulations where we constrained variation in these traits to the population mean. In this context, variation in infectiousness, followed by social aggregation, was the most influential component of transmission. Overall, we show that host heterogeneity in three host traits dramatically affects population-level transmission, but the relative impact of this variation depends on both the susceptible population diversity and the distribution of population-level variation.     

Genetic Variation in Activity of the Enzymes of Glycolysis and Gluconeogenesis between Inbred Strains of Mice

Variation in the activity of 21 liver and 15 erythrocyte enzymes between seven inbred strains of mice has been studied in a single area of metabolism, glycolysis and gluconeogenesis. Most of the variation between the strains is genetic. From the variation within and between inbred strains heritabilities (H²) were determined. Out of 35, 26 showed significant values above 0.4. A comparison with previously published work suggests that enzyme activities have mainly dominance and interaction components of variance, and this is discussed in relation to the variation in quantitative characters such as growth. In nine of the pairwise comparisons of the strains, the activity of the enzyme varied more than two-fold. In these cases the genetics and biochemistry of the enzyme was studied; F₂ progeny were produced and assessed for segregation, and the heat stability of the enzyme was determined. No unequivocal segregation was observed, although in one case we found a considerable difference in heat stability. The variations found were not considered to be great enough to be useful as models of human inborn errors of metabolism or to study metabolic control. If such variants are to be found, sources of variation other than inbred strains must be used.     

Variation among Extracted Lines of Drosophila Melanogaster in Triacylglycerol and Carbohydrate Storage

Whole larvae and whole adult extracts from 26 second chromosome replacement lines of Drosophila melanogaster were analyzed to determine the amounts of stored triacylglycerols and carbohydrates as well as the activities of 13 enzymes in relevant biochemical pathways. Analysis of covariance revealed significant differences among lines in stored lipids and carbohydrates, as well as in activities of most of the enzymes. Significant broad-sense genetic correlations (among adjusted line means) were detected for a number of enzyme pairs. Multiple regression techniques were applied to assess the extent to which the amounts of stored triacylglycerols and carbohydrates could be predicted from the enzyme activities. Significant regressions were found in both adults and larvae, suggesting that modulation of enzyme activities is reflected in different sizes of storage pools. The population genetic consequences of natural selection acting on a phenotype such as energy storage is considered in light of models of metabolic flux in biochemical pathways.     

Post-Translational Modification as a Potential Explanation of High Levels of Enzyme Polymorphism: Xanthine Dehydrogenase and Aldehyde Oxidase in DROSOPHILA MELANOGASTER

Xanthine dehydrogenase (XDH) and aldehyde oxidase (AO) in Drosophila melanogaster require for their activity the action of another unlinked locus, maroon-like (mal). While the XDH and AO loci are on chromosome 3, mal maps to the X chromosome. Although functional mal gene product is required for XDH and AO activity, it is possible to examine the effects of mutant mal alleles in those cases when pairs of mutants complement to produce a partial restoration of activity. To test whether mal mediates a post-translational modification of the XDH and AO proteins, we constructed several mal heteroallelic complementing stocks of Drosophila in which the third chromosomes were co-isogenic. Since all lines were co-isogenic for the XDH and AO structural genes, any variation in these enzymes seen when comparing these stocks must have been produced by post-translational modification by mal. We examined the XDH and AO proteins in these stocks by gel-sieving electrophoresis, a procedure that permits independent characterization of a protein's charge and shape, and is capable of discriminating many variants not detected in routine electrophoresis. In every mal heteroallelic combination, there is a significant alteration in protein shape, when compared to wild type. The magnitude of differences in shape of XDH and AO is correlated both with differences in their enzyme activities and with differences in their thermal stabilities. As the body of this variation appears heritable, any functional differences resulting from these variants are of real genetic and evolutionary interest. A similar post-translational modification of XDH and AO by yet another locus, lxd, was subsequently documented in an analogous manner. The pattern of electrophoretic differences produced by mal and lxd modification is similar to that reported for electrophoretic "alleles" of XDH in natural populations. The implication is that heritable variation in electrophoretic mobility at these two enzyme loci, and potentially at other loci, is not necessarily allelic to the structural gene loci.     

Causes and Consequences of Variation in Energy Storage in Drosophila Melanogaster

Variation in energy storage pools has a proximate cause variation in rates of influx and efflux from the pools, and it may, as an ultimate consequence, result in variation in fitness. The possibility of this chain of causal links motivates attempts to quantify the genetic correlations among pool sizes, enzyme activities and fitness components. In this report, homogenates from 83 second chromosome replacement lines of Drosophila melanogaster were analyzed to determine the amounts of stored triacylglycerols and glycogen as well as the activities of 11 enzymes in relevant metabolic pathways. The viabilities and fecundities of these same lines were determined by a segregation test using the SM5 balancer chromosome. Analysis of covariance revealed significant differences among lines in quantities of stored triacylglycerols and glycogen, as well as in activities of the assayed enzymes. Significant broad-sense genetic correlations were detected for a number of enzyme pairs. Some of the traits showed a significant correlation with viability and fecundity, including lipid and glycogen storage. Multiple regression models that fitted fitness components to linear and quadratic functions of the biochemical traits yielded highly significant fits. The partial regression coefficients indicate the shape of the selection gradient, and instances of significant directional and stabilizing selection were detected.     

Genome-Wide Association Analysis of Tolerance to Methylmercury Toxicity in Drosophila Implicates Myogenic and Neuromuscular Developmental Pathways

Methylmercury (MeHg) is a persistent environmental toxin present in seafood that can compromise the developing nervous system in humans. The effects of MeHg toxicity varies among individuals, despite similar levels of exposure, indicating that genetic differences contribute to MeHg susceptibility. To examine how genetic variation impacts MeHg tolerance, we assessed developmental tolerance to MeHg using the sequenced, inbred lines of the Drosophila melanogaster Genetic Reference Panel (DGRP). We found significant genetic variation in the effects of MeHg on development, measured by eclosion rate, giving a broad sense heritability of 0.86. To investigate the influence of dietary factors, we measured MeHg toxicity with caffeine supplementation in the DGRP lines. We found that caffeine counteracts the deleterious effects of MeHg in the majority of lines, and there is significant genetic variance in the magnitude of this effect, with a broad sense heritability of 0.80. We performed genome-wide association (GWA) analysis for both traits, and identified candidate genes that fall into several gene ontology categories, with enrichment for genes involved in muscle and neuromuscular development. Overexpression of glutamate-cysteine ligase, a MeHg protective enzyme, in a muscle-specific manner leads to a robust rescue of eclosion of flies reared on MeHg food. Conversely, mutations in kirre, a pivotal myogenic gene identified in our GWA analyses, modulate tolerance to MeHg during development in accordance with kirre expression levels. Finally, we observe disruptions of indirect flight muscle morphogenesis in MeHg-exposed pupae. Since the pathways for muscle development are evolutionarily conserved, it is likely that the effects of MeHg observed in Drosophila can be generalized across phyla, implicating muscle as an additional hitherto unrecognized target for MeHg toxicity. Furthermore, our observations that caffeine can ameliorate the toxic effects of MeHg show that nutritional factors and dietary manipulations may offer protection against the deleterious effects of MeHg exposure.     

Gene differences in heading date,height, seed weight and seed yield between two pure line varieties of Triticum aestivum L.

Summary Reciprocal sets of homozygous inbred backcross lines were developed by crossing two pure line varieties (Baart 46 and Ramona) of Triticum aestivum L., followed by two backcrosses to each of the two parent varieties, and six to eight generations of selfing. Data on each inbred backcross line was obtained from twelve plots (from replications in three years). Five genes were responsible for over 95% of the genetic variation for heading date. These genes had pleiotropic effects on plant height that were proportional to their effects on heading date. Two additional genes had detectable effects on plant height. The genes with a measurable effect on height accounted for 90% of the genetic variation in the Baart 46 genetic background. One gene affected seed weight. In the Ramona background, this gene accounted for 80% of the genetic variation in seed weight and 16% of the genetic variation in seed yield. Two genes, responsible for the earliest and latest heading date classes, had large pleiotropic effects on seed yield. They accounted for 60% of the genetic variation in yield. One gene, with no effect on heading date, caused a detectable reduction in yield of 23% in the Baart 46 inbred backcross lines. This gene had no apparent effect in the Ramona genetic background. Quantitative trait genes are sparsely distributed in the genome: fewer than one in four chromosome arms carries a gene with a detectable effect. Gene effects on quantitative traits are not small and similar. The distribution of 22 gene effects for heading date and height is slightly skewed to the right: as the magnitude of effect increases, the frequency of genes having the effect decreases.     

Models of Quantitative Variation of Flux in Metabolic Pathways

As a model of variation in a quantitative character, enzyme activity variation segregating in a population is assumed to affect the flux in simple metabolic pathways. The genetic variation of flux is partitioned into additive and nonadditive components. An interaction component of flux variance is present because the effect of an allelic substitution is modified by other substitutions which change the concentrations of shared metabolites. In a haploid population, the the proportion of interaction variance is a function of the gene frequencies at the loci contributing to the flux variation, enzyme activities of mutant and wild type at variable loci and activities at nonvariable loci. The proportion of interaction variance is inversely related to the ratio of mutant to wild-type activities at the loci controlling the enzyme activities. The interaction component as a function of gene frequencies is at a maximum with high mutant allele frequencies. In contrast, the dominance component which would apply to a diploid population is maximal as a proportion of the total when mutant alleles are at low frequencies. Unless there are many loci with large differences in activity between the alleles, the interaction component is a small proportion of the total variance. Data on enzyme activity variation from natural and artificial populations suggest that such variation generates little nonadditive variance despite the highly interactive nature of the underlying biochemical system.     

Stage-Specific Effects of Candidate Heterochronic Genes on Variation in Developmental Time along an Altitudinal Cline of Drosophila melanogaster

Background

Previously, we have shown there is clinal variation for egg-to-adult developmental time along geographic gradients in Drosophila melanogaster. Further, we also have identified mutations in genes involved in metabolic and neurogenic pathways that affect development time (heterochronic genes). However, we do not know whether these loci affect variation in developmental time in natural populations.

Methodology/Principal Findings

Here, we constructed second chromosome substitution lines from natural populations of Drosophila melanogaster from an altitudinal cline, and measured egg-adult development time for each line. We found not only a large amount of genetic variation for developmental time, but also positive associations of the development time with thermal amplitude and altitude. We performed genetic complementation tests using substitution lines with the longest and shortest developmental times and heterochronic mutations. We identified segregating variation for neurogenic and metabolic genes that largely affected the duration of the larval stages but had no impact on the timing of metamorphosis.

Conclusions/Significance

Altitudinal clinal variation in developmental time for natural chromosome substitution lines provides a unique opportunity to dissect the response of heterochronic genes to environmental gradients. Ontogenetic stage-specific variation in invected, mastermind, cricklet and CG14591 may affect natural variation in development time and thermal evolution.     

Genetic Analysis of Central Carbon Metabolism Unveils an Amino Acid Substitution That Alters Maize NAD-Dependent Isocitrate Dehydrogenase Activity

Background

Central carbon metabolism (CCM) is a fundamental component of life. The participating genes and enzymes are thought to be structurally and functionally conserved across and within species. Association mapping utilizes a rich history of mutation and recombination to achieve high resolution mapping. Therefore, applying association mapping in maize (Zea mays ssp. mays), the most diverse model crop species, to study the genetics of CCM is a particularly attractive system.

Methodology/Principal Findings

We used a maize diversity panel to test the CCM functional conservation. We found heritable variation in enzyme activity for every enzyme tested. One of these enzymes was the NAD-dependent isocitrate dehydrogenase (IDH, E.C. 1.1.1.41), in which we identified a novel amino-acid substitution in a phylogenetically conserved site. Using candidate gene association mapping, we identified that this non-synonymous polymorphism was associated with IDH activity variation. The proposed mechanism for the IDH activity variation includes additional components regulating protein level. With the comparison of sequences from maize and teosinte (Zea mays ssp. Parviglumis), the maize wild ancestor, we found that some CCM genes had also been targeted for selection during maize domestication.

Conclusions/Significance

Our results demonstrate the efficacy of association mapping for dissecting natural variation in primary metabolic pathways. The considerable genetic diversity observed in maize CCM genes underlies heritable phenotypic variation in enzyme activities and can be useful to identify putative functional sites.     

Characterization of cultured tobacco cell lines resistant to ethionine, a methionine analog

Two cultured tobacco cell lines (Nicotiana tabacum L. cv Xanthi) were selected for resistance to growth inhibition by the methionine analog ethionine. Comparison of the free amino acid pool levels in these lines with those of the ethionine-sensitive parental line showed substantial accumulation of methionine (110×), threonine (18×), and lysine (5×). In vitro enzymic analysis of lysine-sensitive aspartate kinase activity showed the resistant lines to contain 16 times that found in the sensitive line. The lysine-sensitive enzymes from both resistant and sensitive lines coeluted from DEAE-cellulose and exhibited similar K_m values. Both showed identical lysine plus S-adenosylmethionine inhibition profiles suggesting that the elevated activity in the resistant lines is not due to a structural change in the lysine-sensitive enzyme but possibly to the level of its expression.